Trichloroethylene (TCE) is widely used as a solvent and a degreasing agent. It has been spilled in the environment and causes great concern because of its toxicity and possible carcinogenic properties. No microorganisms that can grow on this compound as an electron donor are known, but cometabolic conversion of TCE induced by oxygenases has been extensively studied. Several compounds like phenol, toluene, methane, propane, and butane have been demonstrated to produce enzymes that cometabolize TCE. Some of these compounds have been shown to induce the corresponding monooxygenase enzymes that are responsible for the oxidation of the TCE.
Similarly, cis-dichloroethylene (cis-DCE), which is frequently the anaerobic transformation product of TCE, accumulates in the environment and poses an equally important health hazard. The aerobic cometabolism of cis-DCE has been reported to occur with many microorganisms that also degrade TCE. The pathways of degradation for both cis-DCE and TCE are similar.
The aerobic organisms that are capable of oxidizing cis-DCE and TCE possess an oxygenase enzyme for the initial oxidation with growth substrates such as methane and propane, for example. The mechanism of monooxygenase catalyzed oxidation of cis-DCE and TCE is presented in Schemes 1 and 2, respectively. 
The oxidative dechlorination of cis-DCE and TCE requires nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) as the reducing energy source and molecular oxygen as an electron acceptor. The oxidation of the chlorinated ethenes causes the depletion of NADPH in cells. Therefore, NADPH has to be supplied via cometabolic substrate oxidation and endogenous energy reserves. The oxidation of cis-DCE and TCE lead to the formation of the corresponding epoxides, which are highly unstable with half lives of 70 hours to a few seconds, respectively.
Most chlorinated ethenes cannot be utilized as the primary substrate for growth by aerobes, but can be reductively dechlorinated by natural microbial communities and mixed microbial enrichment cultures under anaerobic conditions. For example, the reductive dechlorination of TCE occurs via cis-dichloroethylene (cis-DCE) and vinyl chloride (VC) to ethene by the sequential replacement of the chlorine atoms with hydrogen.
The chlorinated ethenes serve as the electron acceptors in reductive dechlorination. Each dechlorination step requires two electrons, and therefore an electron donor is necessary to complete the reaction. A number of organic compounds have been successfully used as electron donors for the anaerobic dechlorination of TCE. TCE has been shown to be converted to cis-DCE with additions of lactate, propionate, crotonate, butyrate, or ethanol as an electron donor in soil microcosms. Other electron donors such as acetate, glucose, formate, and methanol have also proved to be effective.
Even though a wide range of electron donors support dechlorination, recent studies have shown that the hydrogen produced from the fermentation of these electron donors may be the ultimate electron donor. Cultures enriched with methanol have been shown to dechlorinate using hydrogen as the sole electron donor. Also, it has been shown that a strain of bacteria can grow on tetrachloroethylene (PCE) and hydrogen to produce ethylene. See, for example, DiStefano et al., Appl. Environ. Microbiol., 57, 2287-2292 (1991); Holliger et al., Appl. Environ. Microbiol., 59, 2991-2997 (1993); DiStefano et al., Appl. Environ. Microbiol., 58, 3622-3629 (1992); Maymo-Gatell et al., Appl. Environ. Microbiol., 3928-3933 (1995); Mohn et al., Microbial Reviews, 56, 482-507 (1992); Maymo-Gatell et al., Science, 276, 1568-1571 (1997); and Fennell et al., Environ. Sci. Technol., 31, 918-926 (1997). Even though H2 is not always the only electron donor, it is in many cases the electron donor used by dechlorinators. In natural environments, most H2 becomes available to hydrogenotrophic microorganisms through the fermentation of more complex substrates by other members of the microbial consortium. The dechlorinators then face severe competition from other microorganisms such as sulfate reducers and methanogens for the evolved H2.
Recent work on the anaerobic transformation of chlorinated solvents such as PCE and TCE has centered on determining what substrates can be slowly fermented to deliver a long term source of hydrogen. The goal is to maintain a constant delivery of low concentration of hydrogen, so that the hydrogen is directed towards the dehalogenation process, and not the competing reaction of carbon dioxide with hydrogen to form methane. Thus, the use of slow release substrates hold enormous promise for the implementation of this technology.
Another potential use of slow release substrates is to deliver aerobic substrates to drive the cometabolism of chlorinated solvents. For example, phenol has been demonstrated to be an effective substrate to drive the aerobic cometabolism of TCE; however, direct addition of phenol is problematic because it is a regulated compound and phenol competitively inhibits the enzyme required for TCE cometabolism.
Anaerobic dehalogenation processes are also important for the treatment of chlorinated phenols, such as pentachlorophenol and polychlorinated biphenyls (PCBs). Thus, there is a need for substrates that slowly release compounds that can promote aerobic and/or anaerobic dehalogenation of environmental contaminants, particularly halogenated organic compounds.
The present invention provides a bioremediation method of degrading one or more environmental contaminants in a sample containing microorganisms. In one embodiment, the method includes: determining the presence of the one or more environmental contaminants; and contacting the sample with at least one slow release compound having at least one hydrolyzable organic group capable of forming at least one alcohol, at least one organic acid, or a combination thereof, upon hydrolysis; wherein the slow release compound having at least one hydrolyzable organic group is provided in an amount sufficient to form at least one alcohol, at least one organic acid, or a combination thereof, in an amount sufficient for the microorganisms to degrade the one or more environmental contaminants.
Preferably, determining the presence of one or more environmental contaminants includes measuring the initial concentration of a contaminant and monitoring its degradation into transformation products. Preferably, monitoring degradation of an environmental contaminant includes measuring its concentration or the concentration of its transformation products during the degradation of the environmental contaminant.
In another embodiment, the present invention provides a bioremediation method of degrading a halogenated organic compound in an environmental sample. The method includes: determining the presence of the halogenated organic compound; and contacting the environmental sample with at least one organosilicon compound having at least one hydrolyzable organic group in the presence of at least one type of microorganism.
The environmental contaminant can be selected from the group of organic compounds (e.g., halogenated organic compounds, polycyclic aromatic hydrocarbons, and nitrogenated organic compounds), metals, metal-containing compounds, and mixtures thereof. The compound having at least one hydrolyzable organic group is selected from the group of an organometallic compound, an organophosphorus compound, an organic compound, and mixtures thereof. The bioremediation method can occur under aerobic conditions, anaerobic conditions, or a combination of anaerobic and aerobic conditions. Thus, the microorganisms can be aerobic or anaerobic microorganisms.
The sample containing one or more environmental contaminants can include soil, sediment, sludge, water, or combinations thereof, and the bioremediation method can be carried out in situ. Thus, if the sample is in a subsurface environment, such as groundwater, the contacting step can include injecting a liquid into the subsurface environment using, for example, drive-point devices. Alternatively, recirculation wells can be used. If desired, the sample can be treated ex situ in a bioreactor, for example.
The present invention also provides a kit for bioremediation of an environmental contaminant in a sample comprising at least one type of microorganism. The kit includes: at least one slow release compound having at least one hydrolyzable organic group capable of forming at least one alcohol, at least one organic acid, or a combination thereof, upon hydrolysis; and instruction means for applying the compound to the sample. Preferably, the kit includes a container that allows slow release of the compound having at least one hydrolyzable organic group, the alcohol or organic acid, or combinations thereof.